Technical Abstract:
Certain strains of the ruminant anaerobic bacterium Selenomonas ruminantium have been shown to enhance utilization of xylooligosaccharides under fermentation conditions. Preparations of S. ruminantium beta-D-xylosidase were shown to act on 4-nitrophenyl-beta-D-xylopyranoside and 4-nitrophenyl-alpha-L-arabinofuranoside with a 10-fold preference of the former substrate over the latter. Moreover, natural oligosaccharides, produced from reacting oatspelt xylan and wheat arabinoxylan with a xylanase, were accepted as substrates by the enzyme. Recently, X-ray structures of beta-D-xylosidase belonging to glycoside hydrolase family 43 have been reported for the enzyme from different species. The amino acid sequence of the xylosidase from Clostridium acetobutylicum has 72% identity with that of the S. ruminantium enzyme, and its X-ray structure was used to model the active site of the S. ruminantium enzyme. Beta-xylosidase from S. ruminantium, heterologously expressed in Escherichia coli, was purified to homogeneity for structure and function studies. Steady-state kinetic studies determined that the xylosidase is highly active on natural and artificial substrates. Substrate specificities, reaction stereochemistry, and effects of pH, temperature, and site-directed mutations were determined for the enzyme.